Methods for introducing a gene into an animal cell are roughly classified into physiochemical methods and biological methods. Examples of physiochemical methods include methods such as calcium phosphate coprecipitation, DEAE dextran, lipofection, microinjection, and electroporation. Examples of biological methods include methods using viral vectors.
A viral vector method is a method in which a gene is introduced by utilizing the cell invasion mechanism of virus, that is, an infecting ability.
Virus-derived structural proteins (nucleocapsid, envelope protein, etc.) exist on the surface of a recombinant viral particle prepared by using a viral vector, which have a mechanism for efficient introduction of a gene so that the virus can infect a cell via a receptor on the cell surface. Therefore, a recombinant viral particle prepared using such a viral vector can be used not only to introduce a gene into an animal cell to generate a cell expressing the gene of interest, but also to perform gene therapy, construct a transgenic animal, and so forth.
Viral vectors are grouped into retrovirus vectors, DNA virus vectors, and RNA virus vectors and characterized by the length of a gene that can be introduced, whether the gene is incorporated into the chromosomal genome in a cell, whether the gene can only be introduced into a dividing cell or can also be introduced into a nondividing cell, types of cells that can be infected, cytotoxicity, gene introduction efficiency, and so forth, which depend on the type of the original virus.
Retroviruses have a plus-strand RNA as a genome. This RNA has properties characteristic to mRNA of eukaryotic cells, specifically, a methylated cap structure at the 5′ end and a polyA tail of about 200 nucleotides at the 3′ end. In an infected cell, this RNA is converted to DNA by reverse transcriptase of the virus. Further, the DNA is incorporated into genomic DNA of the host by actions of enzymes encoded by the viral genes. The incorporated DNA is called provirus. A repetitive sequence (long terminal repeat: LTR) occurs at the either end of a provirus, and viral RNA is synthesized by a promoter existing in this sequence. Viral proteins are translated from the synthesized RNA, and a genome-sized RNA is incorporated into the viral particle, which is released out of the cell as a daughter particle.
The RNA structure required for production of a viral particle includes LTR at either end, a primer-binding site sandwiched therebetween, a packaging signal, and a polypurine signal. These are essential cis factors. On the other hand, genes coding for viral proteins are not essential as cis factors, and replication and production of a particle normally occur once viral proteins are supplied within the infected cell.
Therefore, to produce a recombinant retrovirus, a vector from which genes encoded by the retrovirus, such as gag, pol, and env, are removed and into which a gene of interest to be expressed is inserted instead (referred to as a retrovirus vector) is prepared, and this vector is introduced into a cell in which viral proteins are supplied (usually referred to as a packaging cell) to prepare a retrovirus particle incorporated with a foreign gene (Non-patent Document 1).
Examples of the retrovirus include mouse leukemia virus, feline leukemia virus, baboon type C oncovirus, human immunodeficiency virus, adult T cell leukemia virus, and so forth. Furthermore, examples of those reported as recombinant retrovirus vectors include those based on mouse leukemia virus (Non-patent Document 1), those based on human immunodeficiency virus (Non-patent Document 2), and so forth.
A system for production of a recombinant retrovirus consists of two component units, specifically, a retrovirus vector carrying genetic information (a foreign gene of interest) to be introduced and all factors required for packaging and incorporation of the viral genome in cis (recombinant retrovirus DNA) and a retrovirus packaging cell that supplies viral proteins encoded by the gag, pol, and env genes. The recombinant retrovirus particle cannot be released by a packaging cell alone into which a recombinant vector expressing the gag, pol, and env genes is introduced.
To produce a recombinant retrovirus particle, the gag, pol, and env proteins need to be positioned in trans. Therefore, by introducing a retrovirus vector into a packaging cell into which a recombinant vector expressing the gag, pol, and env genes is introduced, a recombinant retrovirus carrying genetic information held in the above-mentioned vector can be produced. Subsequently, when a cell is infected with these viruses, the retrovirus vector will be incorporated into the chromosomal genome in the cell according to the natural retrovirus life cycle.
Thus, the retrovirus vector method is a system constructed for the purpose of efficient incorporation of a specific DNA into the chromosomal genome of the host. However, since the location of the gene of interest to be inserted cannot be predicted, possibilities cannot be ruled out that normal gene may be damaged by insertion, genes in the vicinity of the insertion site may be activated, and the foreign gene of interest may be overexpressed or underexpressed. To overcome these problems, development of a transient expression system using a DNA virus vector that can be utilized as an extrachromosomal gene was promoted.
A DNA virus vector is a vector derived from a DNA virus. DNA virus carries DNA in its viral particle as genetic information. This DNA is replicated by repetition of a process of producing a complementary strand using its own DNA as a template by host-derived DNA-dependent DNA replication enzymes at least as a part of catalysts. Examples of DNA virus vectors that can be utilized as an extrachromosomal gene include adenoviral vectors.
Human adenovirus has about 36-kb linear double-stranded DNA as a genome, and regions included in this genome are roughly divided into early genes E1, E2, E3, and E4 and late genes L1, L2, L3, L4, and L5. The early genes are primarily involved in virus replication, and the late genes are involved in synthesis of viral structural proteins such as capsid. An adenovirus vector used for introduction of a gene is prepared by replacing the E1 region (divided into E1A and E1B, and all adenovirus promoters are activated by E1A), an early gene, with a desired foreign gene (gene of interest) and proliferated using 293 cells, a cell line that can supply E1A in trans (293 cells express E1A). An adenovirus vector deficient in the E1A region cannot be proliferated in a normal cell, which does not express E1A. Since the E3 region is not essential for propagation of virus, it is often removed to increase the insertion size of a foreign gene. Since adenovirus can package a genome up to 105% of the genome size of a wild type in its capsid, a foreign gene of up to 8.1 kb can be inserted by deleting the E1 and E3 regions (Non-patent Document 3).
An adenovirus vector can introduce a gene into a nongrowing cell or a growing cell (Non-patent Document 4). Therefore, this method is suitable for in vivo gene introduction methods. One of disadvantages of this vector is the generally short gene expression period (in units of week). This is because the adenovirus genome exists only within an extrachromosomal region (episome) and is not replicated or amplified. A second disadvantage is that the adenovirus commonly used at present causes nonspecific inflammatory reactions and intensifies a cell-mediated immune response against the vector itself. Therefore, it is difficult to perform continuous administration in gene therapy (Non-patent Document 5).
Viral vectors based on RNA virus are being developed. RNA virus is replicated by repeating the process of generating a complementary strand using its own RNA as a template by its own RNA-dependent RNA replication enzymes as catalysts.
RNA viruses are classified into minus-strand RNA viruses and plus-strand RNA viruses. Representative examples of minus-strand RNA viruses include influenza virus. The influenza virus genome consists of eight minus-strand RNA segments. When influenza virus infects a cell, gene transcription is initiated by proteins in the influenza particle. First, viral RNA polymerase cleaves mRNA of the host cell at a dozen or so nucleotides from the 5′-end cap structure and utilizes the fragments as primers to elongate the RNA strand (plus strand). Viral proteins are translated from this plus-strand RNA. In the replication process, RNA completely complementary to the viral RNA is synthesized, and progeny virus RNA is amplified using this sequence as a template. Then, the viral RNA is packaged together with viral proteins to form a viral particle.
Therefore, to produce influenza virus in a cell culture system, proteins encoded by influenza virus are expressed by RNA polymerase II promoters such as, for example, CMV and CAG promoters, viral RNA is expressed by RNA polymerase I promoters, promoters without a cap structure and polyA, such as, for example, rRNA gene promoters, and the viral RNA is packaged together with viral proteins in the cell to form a viral particle (Non-patent Document 6). However, the amount of virus to be produced is not specified, and this technique has not been established as a technique that can be utilized in view of production to a satisfactory extent.
Examples of viruses classified as plus-strand RNA viruses include Sindbis virus and hepatitis C virus. The genome RNA of a plus-strand RNA virus also functions as messenger RNA (hereinafter referred to as “mRNA”) at the same time and can produce proteins required for replication and particle formation depending on the translation function of the host cell. In other words, the genome RNA of the plus-strand RNA virus itself has a transmitting ability.
Viral vectors derived from Sindbis virus has a basic structure of the genome RNA from which the structural gene region involved in the virus structure is deleted and in which a gene group for proteins required for virus transcription and replication are retained and RNA in which a desired foreign gene is ligated to the downstream of the transcription promoter. When such RNA or cDNA transcribed to this RNA is introduced into a cell, autonomous replication of RNA vector including the foreign gene and transcription of the foreign gene located downstream of the transcription promoter occur, and a foreign gene product of interest is expressed in the cell. Further, a complex that has an infecting ability but not a transmitting ability can be prepared by allowing a cDNA unit expressing structural genes (helper) and a cDNA unit expressing the above-mentioned RNA vector to coexist in a packaging cell (Non-patent Document 7).
Since Sindbis virus uses 67-kDa high-affinity laminin receptor (LAMR) as a receptor and infects nerve cells in high efficiency, Sindbis virus vector draws attention as a system for introducing a gene specifically to nerves (Non-patent Document 8). However, since it has been shown that infection by Sindbis virus induces apoptosis of the host cell (Non-patent Document 9), toxicity is concerned.
The genome of hepatitis C virus (HCV) is plus-strand single-stranded RNA of about 9600 nucleotides. This genome RNA comprises the 5′ untranslated region (also expressed as 5′NTR or 5′UTR), the translated region including a structural region and a nonstructural region, and the 3′ untranslated region (also expressed as 3′NTR or 3′UTR). Structural proteins of HCV are encoded in the structural region, and multiple nonstructural proteins are encoded in the nonstructural region.
Such structural proteins (Core, E1, E2, and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) of HCV are translated as a continuous polyprotein from the translated region, subjected to limited digestion by protease, released, and produced. Of these structural proteins and nonstructural proteins (i.e., viral proteins of HCV), Core is the core protein. E1 and E2 are envelope proteins. Nonstructural proteins are viral proteins involved in replication of the virus itself. NS2 is known to have a metalloprotease activity, and NS3 is known to have a serine protease activity (⅓ on the side of the N terminus) and a helicase activity (⅔ on the side of the C terminus). Furthermore, it is also reported that NS4A is a cofactor for the protease activity of NS3, and that NS5B has an RNA-dependent RNA polymerase activity.
It has been revealed that HCV is classified into many types depending on the genotype or the serotype. According to the phylogenetic analysis method by Simmonds et al. using nucleotide sequences of HCV strains, which is a currently mainstream HCV genotype classification method, HCV is classified into six types including genotypes 1a, 1b, 2a, 2b, 3a, and 3b, and these are further subdivided into several subtypes. Furthermore, the nucleotide sequences of the full-length genomes of some genotypes of HCV have been determined (Non-patent Documents 10 to 13).
An HCV particle is captured by sulfated polysaccharides on the cell surface, binds to a high-affinity receptor via envelope proteins, and is taken up into the endosome by endocytosis. Then, the virus membrane and the endosome membrane fuse, and the nucleocapsid invades the cytoplasm. Translation of the naked viral genome is initiated by Internal Ribosome Entry Site (IRES). Translation and cleavage of a protein occur on the endoplasmic reticulum membrane. The Core protein, the E1 and E2 proteins, and viral RNA replicated on the endoplasmic reticulum are assembled to form a viral particle. Then, the particle buds into the endoplasmic reticulum lumen. It is thought that the particle that has budded is released out of the cell through the Golgi apparatus.
Recently, preparation of an HCV subgenomic RNA replicon as HCV-derived RNA having an autonomous replication ability (Patent Documents 1 and 2 and Non-patent Documents 14 to 16) has enabled analysis of the HCV replication mechanism using cultured cells. This HCV subgenomic RNA replicon is obtained by replacing the structural proteins existing downstream of HCV IRES in the 5′ untranslated region of HCV genomic RNA with the neomycin resistance gene and EMCV-IRES ligated to the downstream thereof. It has been demonstrated that, when introduced into human liver cancer cell Huh7 and cultured in the presence of neomycin, this RNA replicon autonomously replicates in the Huh7 cell. Furthermore, it has been demonstrated that some HCV subgenomic RNA replicons autonomously replicate not only in Huh7 but also in cells such as human cervical cancer cell HeLa or human liver cancer cell HepG2 (Patent Document 3). Furthermore, Patent Document 2 proposes production of HCV virus particle utilizing the full-length HCV genome when a recombinant HCV is used as a vector for gene therapy.    [Patent Document 1] JP Patent Publication (Kokai) No. 2002-171978 A    [Patent Document 2] JP Patent Publication (Kokai) No. 2001-17187 A    [Patent Document 3] International Patent Publication WO2004/104198    [Non-patent Document 1] Mann, R. et al., Cell, 33 (1983) p 153-59    [Non-patent Document 2] Simada, T et al., J Clin Invest. 88 (1991) p 1043-47    [Non-patent Document 3] Betta, A et al., Proc. Natl. Acad. Sci. USA 91 (1994) p 8802-06    [Non-patent Document 4] Burden, S & Yarden, Y., Neuron, 18 (1997) p 847-55    [Non-patent Document 5] Crystal, R. G Science, 270, (1995) p 404-10    [Non-patent Document 6] Neumann, G. & Kawaoka, Y., Virology 287 (2001) p 243-50    [Non-patent Document 7] Berglund, P et al., Biotechnology, 11 (1993) p 916-920    [Non-patent Document 8] Wang, K. S et al., J. Virol. 66 (1992) p 4992-5001    [Non-patent Document 9] Levine, B. et al., Nature, 361 (1993) p 739-42    [Non-patent Document 10] Simmonds, P. et al., Hepatology, 10 (1994) p 1321-24    [Non-patent Document 11] Choo, Q. L et al., Science, 244 (1989) p 359-362    [Non-patent Document 12] Okamoto, H et al., J. Gen. Virol., 73 (1992) p 673-79    [Non-patent Document 13] Mori, S. et al., Biochem. Biophis. Res. Commun. 183 (1992)    [Non-patent Document 14] Blight et al., Science, 290 (2000) p 1972-74    [Non-patent Document 15] Friebe et al., J. Virol., 75 (2001) p 12047-57    [Non-patent Document 16] Kato, T. et al., Gastroenterology, 125 (2003) p 1808-17